Targeting fibrotic signaling pathways by EGCG as a therapeutic strategy for uterine fibroids

Fibrosis is characterized by excessive accumulation of extracellular matrix, which is a key feature of uterine fibroids. Our prior research supports the tenet that inhibition of fibrotic processes may restrict fibroid growth. Epigallocatechin gallate (EGCG), a green tea compound with powerful antioxidant properties, is an investigational drug for uterine fibroids. An early phase clinical trial showed that EGCG was effective in reducing fibroid size and its associated symptoms; however, its mechanism of action(s) has not been completely elucidated. Here, we probed effects of EGCG on key signaling pathways involved in fibroid cell fibrosis. Viability of myometrial and fibroid cells was not greatly affected by EGCG treatment (1–200 µM). Cyclin D1, a protein involved in cell cycle progression, was increased in fibroid cells and was significantly reduced by EGCG. EGCG treatment significantly reduced mRNA or protein levels of key fibrotic proteins, including fibronectin (FN1), collagen (COL1A1), plasminogen activator inhibitor-1 (PAI-1), connective tissue growth factor (CTGF), and actin alpha 2, smooth muscle (ACTA2) in fibroid cells, suggesting antifibrotic effects. EGCG treatment altered the activation of YAP, β-catenin, JNK and AKT, but not Smad 2/3 signaling pathways involved in mediating fibrotic process. Finally, we conducted a comparative study to evaluate the ability of EGCG to regulate fibrosis with synthetic inhibitors. We observed that EGCG displayed greater efficacy than ICG-001 (β-catenin), SP600125 (JNK) and MK-2206 (AKT) inhibitors, and its effects were equivalent to verteporfin (YAP) or SB525334 (Smad) for regulating expression of key fibrotic mediators. These data indicate that EGCG exhibits anti-fibrotic effects in fibroid cells. These results provide insight into mechanisms behind the observed clinical efficacy of EGCG against uterine fibroids.

Uterine fibroids (also known as leiomyomas) are the most common benign tumors of the uterus. Fibroids are highly prevalent in African American women compared to Caucasians. Fibroids are present in approximately 80% of black women and nearly 70% of white women by age of 50 1,2 . Although the majority of fibroids are asymptomatic, nearly 25% women experience significant clinical symptoms. Associated symptoms include heavy and abnormal uterine bleeding, pelvic pain or pressure, infertility or reproductive dysfunction 2,3 . Medical treatments have been approved for treatment of fibroids; however, many treatments are partially effective or are associated with side effects 4 . For example, ulipristal acetate is effective in reducing fibroid size and associated symptoms 5 . However, concerns about the risk of rare but serious liver injury with ulipristal acetate treatment have been raised 6 . Recently, elagolix 7 and relugolix 8 have been shown to be effective to reduce heavy menstrual bleeding in women with uterine fibroids. These treatments are associated with hypoestrogenic effects (especially decreases in bone mineral density). While these may be mitigated by using addback therapy 7,8 , they are not approved for long-term use exceeding 2 years. Surgery is the alternative options for women with symptomatic fibroids. But loss of fertility and surgery-associated adverse effects have a negative impact on women's quality of life. Moreover, the annual cost associated with fibroid management is significant as it is estimated to be between $5.9 billion and $34.4 billion in the United States alone 9 .
Fibroids are composed of an increased mass of extracellular matrix (ECM), characteristic of their fibrotic nature 10 . This excessive accumulation of ECM, may be triggered, at least in part, by an inflammatory response, tissue injury, and angiogenesis 11 . Growth factors and cytokines play an important role in promoting pathological fibrosis 11,12 . It has been shown that TGF-β members (such as TGF-β and activin A) can increase the production of ECM proteins in uterine fibroids [13][14][15] , suggesting their critical role in fibrosis. PAI-1 (plasminogen activator inhibitor 1) and CTGF (connective tissue growth factor) are known to be downstream targets of TGF-β/activin

Effect of EGCG on cell viability and cyclin D1 expression in myometrial and uterine fibroid cells.
An MTS assay was used to assess the effects of EGCG on cell viability of myometrial and uterine fibroid cells. Three matched myometrial and fibroid cell types were used for this study. These included P51 myometrial and fibroid cells, P57 myometrial and fibroid cells, and primary cultures of myometrial and fibroid cells. Cells were treated with EGCG at various concentrations (1, 10, 50, 100 and 200 µM) for 24 h. The percentage absorbance curves show the differential effect of EGCG on cell viability of P51 myometrial and fibroid cells. We found that viability of P51 fibroid cells was reduced by 5% at 100 µM and 28% at 200 µM of EGCG, compared to control (100%), and cells were unaffected by low doses (1-50 µM) (Fig. 1A). On the other hand, normal P51 myometrial cell viability was increased at 1-100 µM and only decreased at 200 µM (19% reduction) (Fig. 1A). For P57 and primary myometrial and fibroid cells, cell viability was not greatly altered by EGCG treatment (Fig. 1B-C). Based on the viability curves and a previous report 24 , we selected the dose of EGCG (100 µM) for the next set of experiments. Cyclin D1 (CCND1) is a critical protein involved in the cell cycle progression. We measured both mRNA and protein levels of cyclin D1 in myometrial and fibroid cells. We found that mRNA levels of CCND1 were increased in P51 fibroid cells by 2.8-fold (Fig. 1D), in P57 fibroid cells by 1.6-fold (Fig. 1E), and in primary fibroid cells by 1.9-fold ( Fig. 1F), compared to respective patient matched myometrial cells. EGCG treatment significantly reduced mRNA levels of CCND1 by 39% in P51 fibroid cells, by 36% in P57 fibroid cells, and by 63% in primary fibroid cells, compared to untreated control fibroid cells (100%) (Fig. 1D-F). Similar to transcript levels, protein levels of cyclin D1 were significantly reduced by EGCG treatment in all three matched myometrial and fibroid cell lines ( Fig. 1G-I). The maximum reduction (80%) was observed in P51 fibroid cells (Fig. 1G), followed by 71% in primary fibroid cells (Fig. 1I), and 66% in P57 fibroid cells (Fig. 1H) following EGCG treatment, compared to control (100%). The EGCG effects on cyclin D1 were not significant in P51myometrial cells (Fig. 1G) and in primary myometrial cells (Fig. 1I), but were significant in P57 myometrial cells (Fig. 1H). Overall, the results of transcript and protein levels of cyclin D1 suggest an antiproliferative effect of EGCG in uterine fibroid cells.

EGCG treatment reduced expression of ECM proteins in uterine fibroid cells.
Since functionally important ECM proteins are critical to fibrosis, we quantified mRNA and protein levels of such proteins, including fibronectin (FN1). Fibronectin is known to be overexpressed in uterine fibroids 29 . As expected, we also found that the mRNA levels of FN1 were higher in P51 fibroid cells by 2.5-fold ( Fig. 2A), in P57 fibroid cells by 2.2-fold (Fig. 2B), in primary fibroid cells by 2.6-fold (Fig. 2C), compared to myometrial cells, which were significantly reduced by EGCG treatment as by 39%, 59%, and 55%, respectively, compared to respective control (100%). While the mRNA levels of fibronectin were unaffected by EGCG treatment in primary myometrial cells (Fig. 2C), levels were reduced by EGCG treatment in P51 and P57 myometrial cells ( Fig. 2A-B). Similar to mRNA levels, the protein levels of fibronectin were also higher (1.5 to 2.2-fold) in all three sets of fibroid cells, compared to respective myometrial cells (Fig. 2D-F). EGCG treatment significantly reduced fibronectin protein levels by 46-52% in fibroid cells, compared to untreated control fibroid cells (100%) (Fig. 2D-F). Fibronectin protein levels were not significantly reduced in P51 and P57 myometrial cells (Fig. 2D-E) but were significant in primary myometrial cells (Fig. 2F). Our extended experiment examined the effect of EGCG on the expression of collagens, which are major components of fibroid ECM. Real time qPCR data showed that COL1A1 (collagen type I alpha 1 chain) mRNA levels were 1.64-fold higher in P57 fibroid cells, and 2.6-fold higher in  Fig. 1A-B). Overall, these results suggest that EGCG exhibited a differential effect on fibroid compared to myometrial cells and revealed antifibrotic effects as evident by inhibition of fibronectin and collagen production in fibroid cells. show protein quantification. Membranes were cut into several pieces (based on the molecular weight of proteins of interest) prior to hybridization with primary antibodies during blotting. Data are presented as mean ± SEM of two to four independent experiments. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001. www.nature.com/scientificreports/ EGCG treatment reduced expression of downstream and upstream mediators of fibrosis in uterine fibroid cells. Next, we focused on mRNA and protein levels of PAI-1 and CTGF in response to EGCG treatment. In a previous study, we reported that PAI-1 and CTGF are overexpressed in uterine fibroid cells 20 . Here we found that the mRNA levels of PAI-1 were 4.1-fold higher in P51 fibroid cells (Fig. 3A), 1.13-fold in P57 fibroid cells (Fig. 3B), and 1.4-fold in primary fibroid cells (Fig. 3C), compared to myometrial cells. EGCG treatment significantly reduced (46-57%) mRNA levels of PAI-1 across all three sets of fibroid cells, compared to untreated control fibroid cells (100%) (Fig. 3A-C). Western blot analysis showed that the protein levels of PAI-1 were higher (2.3-fold) in primary fibroid cells, compared to myometrial cells ( Fig. 3F) but not in immortalized P51 and P57 fibroid cells ( Fig. 3D-E). We observed that EGCG greatly reduced (66-76%) protein levels of PAI-1 in all three fibroid cells, compared to untreated control fibroid cells (100%) (Fig. 3D-F). Next, we quantified the expression levels of CTGF in fibroid and myometrial cells after treatment with EGCG. We found that the basal levels of CTGF mRNA were higher (2.7-3.9-fold) in all three fibroid cells, compared to myometrial cells ( Fig. 4A-C). EGCG treatment significantly reduced (36%) mRNA levels of CTGF in P57 fibroid cells (Fig. 4B), but not in P51 fibroid cells (Fig. 4A), or in primary fibroid cells (Fig. 4C), compared to untreated control fibroid cells (100%). We found higher levels of CTGF protein in P51 fibroid cells (1.63-fold) (Fig. 4D), in P57 fibroid cells (1.64-fold) (Fig. 4E), and primary fibroid cells (2.33-fold) (Fig. 4F). EGCG treatment induced a significant reduction in protein levels of CTGF in all three sets of fibroid cells (Fig. 4D-F). The maximum reduction of CTGF protein levels by EGCG was 86% in P51 fibroid cells (Fig. 4D), followed by 77% in P57 fibroid cells (Fig. 4E), and 66% in primary fibroid cells (Fig. 4F), compared to untreated control fibroid cells (100%). Notably, effects of EGCG on CTGF protein levels were not significant in P51 myometrial and primary myometrial cells but they were statistically significant in P57 myometrial cells ( Fig. 4D-F). Membranes were cut into several pieces (based on the molecular weight of proteins of interest) prior to hybridization with primary antibodies during blotting. NT = untreated control cells (not exposed to EGCG). Data are presented as mean ± SEM of two to four independent experiments. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001. www.nature.com/scientificreports/ In addition to CTGF and PAI-1, α-SMA is also considered as an important mediator in fibrosis 30 . The expression of α-SMA is considered a functional marker for myofibroblasts, which contribute to fibrosis 31,32 . As an upstream target of CTGF, PAI-1 and α-SMA 33-35 , we also included profibrotic growth factors activin A (INHBA) and TGF-β (TGFB) in our studies. An active TGF-β1 plays important role in converting fibroblasts into contractile myofibroblasts 36 . Experiments revealed that mRNA levels of α-SMA (ACTA2) were 3.8-fold higher in P57 fibroid cells, which was reduced by 69% with EGCG treatment, compared to control (100%) (Supplementary Fig. 2A). Western blot analysis showed that the protein levels of α-SMA were higher (4.9-fold) in P57 fibroid cells, compared to P57 myometrial cells ( Supplementary Fig. 2B). EGCG treatment severely reduced (71%) α-SMA protein expression in P57 fibroid cells but not in P57 myometrial cells, compared to control (100%) (Supplementary Fig. 2B). We found higher mRNA levels of INHBA (4.3-fold), TGFB1 (2.0-fold), and TGFB2 (5.1-fold) in P51 fibroid cells, compared to normal myometrial cells ( Supplementary Fig. 2C-E). EGCG treatment significantly reduced mRNA levels of INHBA by 60%, TGFB1 by 40%, and TGFB2 by 50% in P51 fibroid cells, compared to their respective untreated controls (100%) (Supplementary Fig. 2C-E). Overall, these results suggest that fibroid cells highly express several key mediators of fibrosis, which were effectively reduced by treatment with EGCG.

EGCG treatment altered the activation of fibrotic signaling pathways in uterine fibroid cells.
To identify the direct targets of EGCG in uterine fibroid cells, we tested the effects of EGCG on different signaling pathways, including YAP, Smad, β-catenin, JNK, and AKT. The fibrotic role of YAP and Smad has been www.nature.com/scientificreports/ reported in different organs 16,37,38 as well as in uterine fibroids 14,19,20,39 . However, the fibrotic roles of β-catenin, JNK, and AKT are not fully understood in uterine fibroids 16 . Our recent data suggest that Hippo signaling (YAP activation) is involved in producing ECM proteins as well as transcription of profibrotic genes in fibroid cells 20,39 .
To investigate if EGCG might alter Hippo/YAP signaling, we treated fibroid and myometrial cells with EGCG at 100 µM for 24 h. We quantified proteins levels of main transcriptional effector of Hippo/YAP signaling, YAP (non-phospho YAP; transcriptionally active). Western blot analysis showed that the protein levels of non-p-YAP were 2.6-fold higher in P51 fibroid cells, compared to myometrial cells (Fig. 5A), suggesting an activation of YAP signaling in fibroid cells. EGCG selectively reduced protein levels of non-p-YAP by 73% in P51 fibroid cells, compared to untreated control fibroid cells (100%) (Fig. 5A). The protein levels of non-p-YAP were unaffected by EGCG treatment in normal myometrial cells (Fig. 5A). We also found that EGCG treatment regulated BIRC5 expression in fibroid cells. BIRC5 (Baculoviral IAP Repeat Containing 5, also known as survivin) is a YAP-responsive gene 40 , which is known to inhibit apoptosis and promote cell proliferation 41 . We found that the basal mRNA levels of BIRC5 were 1.5-fold higher in P51 fibroid cells, compared to P51 myometrial cells, which was significantly reduced (45%) by EGCG treatment, compared to untreated control (100%) (Fig. 5B). Overall, these results suggests that the antifibrotic effects of EGCG is mediated, at least in part, by alteration of fibrotic signaling involving YAP signaling. As previously reported 14,19 , Smad signaling is involved in mediating fibrotic effects in uterine fibroid cells. To investigate if EGCG could affect Smad signaling, we treated fibroid and myometrial cells with EGCG at 100 µM for 24 h. We quantified proteins levels of main transcriptional effector of Smad signaling, phospho-Smad2 www.nature.com/scientificreports/ (active). Western blot analysis showed that the protein levels of pSmad2 were 1.8-fold higher in P51 fibroid cells, compared to P51 myometrial cells ( Supplementary Fig. 3), suggesting an activation of Smad signaling in fibroid cells. Treatment of P51 fibroid cells with EGCG showed no reduction on pSmad2 levels ( Supplementary Fig. 3).
In contrast, the levels of pSmad2 were significantly increased by EGCG treatment in P51 myometrial cells (Supplementary Fig. 3). Although the role of Smad signaling in fibroid cell fibrosis is well understood, EGCG was not able to alter the activation of this signaling. While the role of β-catenin in the regulation of cell proliferation, differentiation, and apoptosis is well-known, the role of β-catenin in mediating fibrosis in uterine fibroid cells has not been clearly demonstrated 21 . To study whether EGCG could regulate β-catenin signaling, we treated P51 myometrial and fibroid cells with EGCG at 100 µM for 24 h. We quantified proteins levels of the transcriptionally active form of β-catenin (non-phosphoβ-catenin). We found that protein levels of active β-catenin were higher (1.8-fold) in P51 fibroid compared to P51 myometrial cells (Fig. 5C), suggesting an activation of β-catenin signaling in fibroid cells. EGCG treatment decreased β-catenin (active) protein levels by 51% in fibroid cells, compared to untreated control fibroid cells (100%) (Fig. 5C). Notably, in P51 myometrial cells, the protein levels of β-catenin (active) were not significantly affected by EGCG treatment (Fig. 5C). We also quantified the downstream target of β-catenin signaling, MYC (MYC proto-oncogene, bHLH transcription factor). We found that the basal mRNA levels of MYC were 1.6-fold higher in P51 fibroid cells, compared to P51 myometrial cells (Fig. 5D). EGCG treatment reduced MYC transcript levels by 38% in fibroid cells, compared to untreated control fibroid cells (100%) (Fig. 5D). Overall, these results suggest that β-catenin signaling can be targeted by EGCG in fibroid cells.
JNK and AKT signaling pathways are commonly associated with cell growth and survival 42 . To study if EGCG could regulate JNK signaling, we treated P51 myometrial and fibroid cells with EGCG at 100 µM for 24 h. We quantified proteins levels of active form of JNK. We found that protein levels of phospho-JNK (Thr183/Tyr185) (active) were higher (1.9-fold) in P51 fibroid compared to P51 myometrial cells (Fig. 5E), suggesting an activation of JNK signaling in fibroid cells. EGCG treatment significantly reduced phospho-JNK (Thr183/Tyr185) levels by 48% in P51 fibroid cells, compared to untreated control fibroid cells (100%) (Fig. 5E). Remarkably, in P51 myometrial cells, the protein levels of phospho-JNK (Thr183/Tyr185) were not significantly affected by EGCG treatment (Fig. 5E). Overall, these results suggest that EGCG can effectively target JNK signaling.
To study if EGCG could regulate AKT signaling, we treated primary myometrial and fibroid cells with EGCG at 100 µM for 24 h. We quantified proteins levels of active form of AKT. We found that protein levels of phospho-AKT (Ser473) (active) were higher (1.3-fold) in primary fibroid cells, compared to primary myometrial cells (Fig. 5F), suggesting an activation of AKT signaling in fibroid cells. EGCG treatment significantly reduced phospho-AKT (Ser473) levels by 35% in primary fibroid cells but not in primary myometrial cells, compared untreated control (100%) (Fig. 5F). These results suggest that EGCG can target AKT signaling.

Discussion
We report for the first time that EGCG induced antifibrotic effects in uterine fibroid cells. We also found that fibrosis was mediated by multiple signaling pathways, which can be effectively blocked by EGCG treatment (Fig. 6E-F). Previously, EGCG was reported to induce antiproliferation and apoptosis in HuLM (human uterine leiomyoma) cells 23 . The antiproliferative effects of EGCG in HuLM cells were mediated by downregulation of catechol-o-methyltransferase (COMT) activity 24 . Later, its antiproliferative and apoptotic effects were confirmed in rat leiomyoma (ELT3) cells in vitro and in a nude mice model 25 . Zhang et al. reported that consumption of EGCG (1.25 mg EGCG/day for 4 and 8 weeks) by female athymic nude mice showed a significant reduction in the volume and weight of tumors 25 . Similar findings were observed against spontaneous tumors of Japanese quail with 12-month treatment of EGCG (200 or 400 mg of EGCG/kg of diet) 26 . These results of antifibrotic effects and alteration of fibrotic signaling pathways by EGCG in uterine fibroid cells represent an important addition to the understanding of EGCG action.
Previously, the effects of EGCG were studied in only immortalized fibroid cells. Here we included two varieties of immortalized fibroid and myometrial cells 43 www.nature.com/scientificreports/ curves revealed that EGCG differentially affected the cell viability of P51 myometrial and fibroid cells. While P51 myometrial cells grew normally at 1-100 µM of EGCG, cell proliferation of fibroid cells was slightly reduced (5%) at 100 µM and was unaffected at 1-50 µM. The viability of P57 and primary fibroid and myometrial cells was not greatly affected by EGCG. However, the reduction in transcript and protein levels of cyclin D1 (an important regulator of cell cycle progression) by EGCG at 100 µM was evident in P51 fibroid cells. Similar results were found in P57 fibroid cells and primary fibroid cells. These results suggest that EGCG may differentially affect cell www.nature.com/scientificreports/ cycle progression between myometrial and uterine fibroid cells, and may represent a mechanism of the clinical efficacy of EGCG against uterine fibroids. The presence of ECM proteins, such as fibronectin in fibroids, is critical the pathogenesis of the tumors. TGF-β members (such as TGF-β and activin A) have been reported to increase deposition of ECM proteins in uterine fibroids [13][14][15] , which supports their critical role in mediating fibroid cell fibrosis. An ECM-rich rigid structure is thought to contribute to abnormal bleeding in the uterus 10 . Indeed, in a previous study, we reported a decrease in fibroid-related pain in patients treated with collagenase, an antifibrotic compound that specifically degrades type I and type III collagens 45 . Therefore, our approach was to test the antifibrotic effect of EGCG in fibroid cells by analyzing expression of fibronectin and collagen as well as profibrotic growth factors (activin A and TGFβ). Fibronectin and collagen levels were elevated in fibroid cells and were downregulated by EGCG treatment. EGCG treatment also decreased the levels of activin A, TGF-β1, and TGF-β2 in fibroid cells. This result suggests the potential antifibrotic effects of EGCG against uterine fibroid cells, might be mediated by downregulation of activin A/TGF-β-mediated action. Consistent with this observation, we observed that downstream targets of activin A/TGF-β, the PAI-1 and CTGF, were significantly reduced by EGCG in fibroid cells. However, we noted that phospho-Smad2 levels were not reduced following EGCG treatment in leiomyoma cells, suggesting an interesting complexity to EGCG actions. Alpha-SMA is a marker of myofibroblasts 18 . We observed that the levels of α-SMA were higher in fibroid cells, which was severely reduced by EGCG. Overall, these findings suggest that EGCG may exert antifibrotic effects via disruption of key mediators of fibrosis.
Next, we extended experiments to identify the direct targets of EGCG in uterine fibroid cells. We tested the effects of EGCG on five signaling pathways, including YAP, Smad, β-catenin, JNK, and AKT. While YAP and Smad are well-known for their fibrotic role in different organs, including fibroids 16,37,38 . The roles of β-catenin, JNK, and AKT are less studied in this context 16 . We found that uterine fibroid cells highly expressed transcriptional effectors or active proteins of YAP, Smad, β-catenin, JNK and AKT. Treatment of fibroid cells with EGCG showed a significant reduction in protein levels of non-p-YAP (active), non-p-β-catenin (active), phospho-JNK (active), and phospho-AKT (active) but not phospho-Smad2 (active), suggesting an ability of EGCG to regulate fibrosis in fibroid cells through alteration of YAP, β-catenin, JNK and AKT signaling pathways.
Finally, we conducted a comparative study to evaluate the efficiency of EGCG along with synthetic inhibitors in regulating expression of fibrotic mediators (such as fibronectin, PAI-1, CTGF, and α-SMA). Western blots showed that EGCG was more efficacious than ICG-001, SP600125 and MK-2206, and was equally effective with verteporfin and SB525334 in modulating expression of fibrotic mediators. This observation opens the possibility of combination treatment approach. Verteporfin is an FDA approved drug, which is used to eliminate abnormal blood vessels in the eye associated with conditions such as macular degeneration. Our previous reports 20 and current data suggest that the combination treatment of EGCG and verteporfin might be a viable option for future study. SB525334 is also promising and showed tumor specific efficacy in the Eker rat model 19 . However, the therapeutic efficacy of SB525334 has been tempered because of mitogenic and antiapoptotic effects for epithelial cells in the kidney of Eker rats 19 .
In conclusion, we found that EGCG induced antifibrotic effects and altered multiple signaling pathways involved in fibrosis in fibroid cells. These results support further investigation of EGCG as treatment for fibroid growth and fibroid-associated symptoms in clinical studies. To date, one clinical trial shows that EGCG is effective in reducing fibroid volume and fibroid associated symptoms without any adverse events group 27 . Another recent phase I clinical study reported the hepatic safety profile in women with and without fibroids. In this study, no signs of drug induced liver injury was reported in women who took 720 mg of EGCG alone or in combination with clomiphene citrate or letrozole for 5 days 28 . The limited number of clinical data suggest that EGCG is welltolerated and is not associated with liver toxicity. Our data provide insight into the underlying mechanism(s) underlying the observed effects on fibroid growth.
Cell culture. Immortalized human uterine fibroid (P51 and P57) and patient-matched myometrial (P51 and P57) cells were collected from Minnie Malik, PhD and William Catherino M.D., Ph.D.; Uniformed Services University of the Health Sciences, Bethesda, MD). For P51 fibroid and myometrial cells, samples were collected from African American woman (44 years-old) who underwent a hysterectomy because of symptoms of bleeding and cramping. The subject had not taken GnRH analogues, such as leuprolide acetate (Lupron), cetrorelix or elagolix or hormone therapy for 3 months prior to surgery. For P57 fibroid and myometrial cells, samples were collected from an African American woman (45 years-old) who underwent a hysterectomy for heavy menstrual bleeding. The subject had not taken GnRH analogues or had been on hormone therapy for 3 months prior to surgery. No phytoestrogen was reported. Both P51 and P57 cells were immortalized by HPV-16 (Human papillomavirus 16) as previously described 43,44 . Briefly, primary cells were infected at 40-50% confluence on first passage with retrovirus stock (pSLXN virus with geneticin selection gene was a gift from Dr Rhim, Center for Prostrate Disease Research, Bethesda, MD). To enhance infection by the retroviral vector, polybrene (5 μg/mL) was added to each flask. After incubation at 37 °C for 24 h, the cells were washed once with PBS, heated to 37 °C and cultured in fresh DMEM-F12 supplemented with 10% FBS (Invitrogen). The cells were maintained at 37 °C and 5% CO 2 for 48 h before adding fresh media containing 100 μg/mL of geneticin (Sigma-Aldrich  Table 1). RPLP0 was amplified under the same conditions for normalizing quantitative data. The relative mRNA expression was calculated using the ΔΔCT method and is presented as fold increase or decrease relative to control.
Western blot. P51, P57, and primary human uterine fibroid and myometrial cells were seeded onto 100 mm dish and cultured to reach ⁓ 70% confluency, and then serum starved for 24 h. Next day, cells were treated with EGCG at 100 µM for 24 h, and cell were left untreated (control). Cells were washed with PBS and lysed with RIPA buffer (Sigma #R0278) containing protease and phosphatase inhibitor cocktail (Thermo Fisher Scientific). Protein concentrations were quantified using Pierce™ BCA Protein Assay Kit (Thermo Fisher Scientific). An equal volume (30-50 μg) of protein lysates were loaded onto 4-12% NuPAGE gels (Thermo Fisher Scientific), resolved by SDS-PAGE under reducing conditions, and then transferred to 0.2-μm nitrocellulose membranes in an X-cell II apparatus (Thermo Fisher Scientific). Ponceau S solution (Sigma #P7170) was used for the detection of protein on nitrocellulose membranes. Based on the molecular weight of proteins of interest, nitrocellulose membranes were cut prior to hybridization into several pieces. After blocking membranes with 5% non-fat-milk with TBST (1X TBS, 0.1% Tween 20) for 1 h, membranes were incubated overnight at 4 °C with primary antibodies (Supplementary Table 2). Next day, membranes were washed three times (5 min each) with TBST and then incubated with appropriate horseradish Peroxidase (HRP)-conjugated secondary antibodies as 1: 30,000 dilutions (GE Healthcare #NA934V or NA931V) with 5% non-fat-milk with TBST for ⁓ 2 h at room temperature. Membranes were washed three times (5 min each) with TBST and immunoreactive proteins were visualized using a SuperSignal™ West Pico PLUS Chemiluminescent Substrate (Thermo Fisher Scientific) in an Azure Imager c300 system (Azure Biosystems, Dublin, California). The band intensity was quantified using Javabased image processing program, Image J 1.52a and normalized against corresponding anti-β-actin or α-actinin. Since our focus was on proteins involved in fibrosis, and levels of cytoskeletal proteins can vary in response to www.nature.com/scientificreports/ mechanical stimulation, rather than rely solely on β-actin, we also normalized expression to α-actinin. Data are presented as fold increase or decrease relative to control.

Statistical analysis.
Cell viability data are represented as percentage mean ± SEM of 2-3 independent experiments. The fold changed data of mRNA and protein are presented as mean ± SEM of 2-7 independent experiments. The conversion of fold change to percentage was performed using a simple proportion as we did previously 52 . The baseline "fold" expression for control samples was set to "100%". As an example, for controls, it is set at 1*100% = 100%; and if the relative expression of treatment was 0.2, then the percent expression would be 0.2*100% = 20%. Therefore, the percent change in gene or protein expression with respect to control is equal to the treatment minus the control value. Therefore, 20-100% = − 80%. The negative sign denotes a decrease in expression. The Mann-Whitney U test was used to evaluate the differences between treatment and control group. Differences were considered significant at p < 0.05.